Combining Protein Phase Separation and Bio‐orthogonal Linking to Coimmobilize Enzymes for Cascade Biocatalysis

Abstract The designed and ordered co‐immobilization of multiple enzymes for vectorial biocatalysis is challenging. Here, a combination of protein phase separation and bioorthogonal linking is used to generate a zeolitic imidazole framework (ZIF‐8) containing co‐immobilized enzymes. Zn ²⁺ ions induce the clustering of minimal protein modules, such as 6‐His tag, proline‐rich motif (PRM) and SRC homology 3 (SH3) domains, and allow for phase separation of the coupled aldoketoreductase (AKR) and alcohol dehydrogenase (ADH) at low concentrations. This is achieved by fusing SpyCatcher and PRM‐SH3‐6His peptide fragments to the C and N termini of AKR, respectively, and the SpyTag to ADH. Addition of 2‐methylimidazole results in droplet formation and enables in situ spatial embedding the recombinant AKR and ADH to generate the cascade biocalysis system encapsulated in ZIF‐8 (AAE@ZIF). In synthesizing (S)‐1‐(2‐chlorophenyl) ethanol, ater 6 cycles, the yield can still reach 91%, with 99.99% enantiomeric excess (ee) value for each cycle. However, the yield could only reach 72.9% when traditionally encapsulated AKR and ADH in ZIF‐8 are used. Thus, this work demonstrates that a combination of protein phase separation and bio‐orthogonal linking enables the in situ creation of a stable and spatially organized bi‐enzyme system with enhanced channeling effects in ZIF‐8.